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Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The purpose of this study was to investigate the association of the selected MYH9 single nucleotide polymorphisms (SNPs) with chronic kidney disease (CKD) and its related co-morbidities in the South African mixed ancestry population residing in Bellville South, Cape Town. In 2008, two landmark studies identified SNPs in the MYH9 gene which explained most of the increased risk for non-diabetic CKD in African Americans. These polymorphisms were later found to be weakly associated with diabetic nephropathy. Three SNPs that exhibited independent evidence for association with CKD were selected (rs5756152, rs4821480 and rs12107). These were genotyped using a Taqman genotyping assay on a BioRad MiniOpticon and confirmed by sequencing in 724 subjects from Bellville South, Cape Town, South Africa. Prevalent CKD was defined based on the estimated glomerular filtration rate calculated using the modification of diet in renal disease (MDRD) formula. Chronic kidney disease was present in 214 subjects (29.6%), 96.3% were stage 3 and only 8 subjects were stage 4. In additive allelic models, adjusted for age and gender, rs5756152 demonstrated an association with kidney function whereby each G allele of rs5756152 increased eGFR by 3.67 ml/min/1.73, reduced serum creatinine by 4.5% and increased fasting plasma glucose by 0.51 mmol/L. When an interaction model was used, the effect of rs5756152 on serum creatinine, eGFR and blood glucose levels was retained, and enhanced, but only in diabetic subjects. In addition, rs4821480 T allele increased eGFR while rs12107 A allele decreased glucose levels in diabetic subjects. In contrast to reports that MYH9 SNPs are strongly associated with non-diabetic end stage renal disease, our study demonstrated that rs5756152 and rs4821480 are associated with early kidney function derangements in type 2 diabetes whilst rs12107 is associated with glucose metabolism. Our findings, along with previous reports, suggest that the MYH9 gene may have a broader genetic risk effect on different types of kidney diseases than previously thought. / AFRIKAANSE OPSOMMING: Hierdie studie het ondersoek ingestel na die verband tussen drie gekose MYH9-enkelnukleotied-polimorfismes (SNP’s) en chroniese niersiekte (hierna ‘niersiekte’), wat verwante ko-morbiditeite insluit, onder ’n Suid-Afrikaanse populasie van gemengde afkoms in Bellville-Suid, Kaapstad. Twee rigpuntstudies het in 2008 op SNP’s in die MYH9-geen afgekom wat verklaar het waarom Afro-Amerikaners ’n hoër risiko vir niediabetiese niersiekte toon. Later is bevind dat hierdie polimorfismes ook ’n swak verband met diabetiese nefropatie het. Drie SNP’s wat elk onafhanklik bewys gelewer het van ’n verband met niersiekte is vervolgens gekies (rs5756152, rs4821480 en rs12107). Die SNP’s is daarná met behulp van die Taqman-toets op ’n BioRad MiniOpticon aan genotipering onderwerp, en is toe deur middel van reeksbepaling by 724 proefpersone van Bellville-Suid, Kaapstad, Suid-Afrika, bevestig. Die voorkoms van niersiekte is bepaal op grond van die geraamde glomerulêre filtrasietempo (eGFR), wat aan die hand van die ‘niersiekte-dieetveranderings’- (MDRD-)formule bereken is. Daar is bevind dat 214 proefpersone (29,6%) aan chroniese niersiekte ly – 96,3% was in fase 3 en slegs agt proefpersone in fase 4. In toegevoegde alleliese modelle wat vir ouderdom en geslag aangepas is, het rs5756152 ’n verband met nierfunksie getoon: Elke G-allel van rs5756152 het eGFR met 3,67 ml/min/1,73 verhoog, serumkreatinien met 4,5% verlaag en vastende plasmaglukose met 0,51 mmol/L verhoog. Toe ’n interaksiemodel gebruik is, is die effek van rs5756152 op serumkreatinien, eGFR en bloedglukosevlakke behou en versterk, hoewel slegs by diabetiese proefpersone. Daarbenewens het die T-allel van rs4821480 eGFR verhoog, terwyl die A-allel van rs12107 ook glukosevlakke by diabetiese proefpersone verlaag het. In teenstelling met bewerings dat MYH9-SNP’s ’n sterk verband met niediabetiese eindstadiumniersiekte toon, het hierdie studie bewys dat rs5756152 en rs4821480 met vroeë nierfunksieversteurings by tipe 2-diabetes verband hou, terwyl rs12107 weer met glukosemetabolisme verbind word. Tesame met vorige studies, doen hierdie navorsingsbevindinge dus aan die hand dat die MYH9-geen dalk ’n groter genetiese risiko-effek op verskillende tipes niersiekte het as wat voorheen vermoed is. / Cape Peninsula University of Technology Research Fund / University of Stellenbosch Merit Bursary

Female reproductive performance is a critical component of sustainable pig production systems. There is abundant evidence of genetic variation in these traits among pig breeds. The aims of this study were to identify quantitative trait loci (QTL) affecting reproductive traits and to identify and characterise positional candidate gene(s) underlying the QTL. A Large White - Meishan F2 population was scanned for QTL with effects on reproductive traits. This analysis revealed 13 putative QTLs on seven different chromosomes with effects on five different traits: ovulation rate (OR), teat number (TN), prenatal survival (PS), total born alive (TBA) and litter size (LS). QTL for PS and LS on chromosome 8 were fine mapped and Secreted Phosphoprotein 1 (SPP1) confirmed as a candidate gene. A genome-wide association study was performed on a diverse population of different breeds and crosses lines, for reproductive traits including LS, TBA, number of stillborn piglets, and number of mummified piglets. Fourteen SNPs were found significantly associated with reproductive traits. The functional study of SPP1 examined the hypothesis that differences in foetal growth may be associated with the effectiveness of conceptus attachment, as measured by SPP1 expression. Patterns of SPP1 mRNA and protein expression in placental and uterine tissues supplying the smallest and a normal-sized foetus from the same uterus were examined in Large White-Landrace (LW-LR), Large White (LW) and Meishan (MS) females 40 and 45 of pregnancy. The smallest LW-LR foetuses tended to have a higher level of SPP1 mRNA in endometrium tissue compared to the normal-sized foetuses. However, placenta expression was higher in the normal-sized foetuses compared to the smallest ones. SPP1 protein levels in normal sized foetuses were significantly higher than in the smallest litter mates for all the tissues. Significantly higher levels of SPP1 mRNA and protein were found in MS compared to LW. In both breeds, significant differences between sizes were found in some tissues, with similar expression patterns in respect to size, for both mRNA and protein in endometrial tissues when compared to contemporary LW. In placenta, the direction of the expression differed between breeds, with a higher expression of mRNA and protein in the normal-sized MS foetuses and in the smallest sized LW foetuses. The comparison of SPP1 expression between different foetal sizes and different breeds revealed associations between breed, foetal size, and SPP1 protein, factors implicated in PS and LS. These results together with the genetic evidence indicate that the potential role of SPP1 in placental and foetal development merits further investigation.

O eixo hipotálamo-pituitária-adrenal é o principal sistema neuroendócrino envolvido na regulação e adaptação da resposta ao estresse e, o principal hormônio secretado é o cortisol. O cortisol exerce seus efeitos por meio dos receptores mineralocorticoide (MR) e glicocorticoide (GR). Variações nos genes desses receptores têm sido associadas à sensibilidade aos glicocorticoides e mudanças no perfil metabólico. O objetivo geral desse trabalho foi compreender a variabilidade existente em relação às respostas fisiológicas de bovinos por meio da identificação de polimorfismos genéticos em genes envolvidos na resposta ao estresse e, verificar as consequências dessa variação genética em características de qualidade da carne. Dessa forma, três abordagens foram propostas: (1) avaliar a incidência de carne DFD (dark, firm and dry) e seu impacto no perfil metabólico, endócrino e características de qualidade da carne bovina, uma vez que o estresse é um dos principais fatores que levam a essa condição desfavorável; (2) avaliar a contribuição de fatores genéticos, por meio da identificação de polimorfismos de nucleotídeo único (SNPs), no gene do MR e GR, e suas associações com as características mensuradas; (3) avaliar os efeitos desses polimorfismos sobre o perfil proteico do músculo bovino. Foram utilizados 241 bovinos da raça Nelore. Os resultados evidenciaram implicações direta do pH 24 horas post-mortem nos atributos de cor e perdas por cozimento da carne. A incidência de carnes DFD (pH>=5,8) foi de 18,7%. Os polimorfismos identificados mostraram influenciar em algumas características mensuradas. Os SNPs NR3C2_1 e NR3C2_2 no gene do MR foram associados ao conteúdo de glicogênio muscular e nível plasmático do hormônio adrenocorticotrófico (ACTH) post-mortem, e o SNP NR3C1_1 no gene do GR foi associado aos níveis plasmático de cortisol post-mortem. As análises proteômicas demonstraram que a maioria das proteínas reguladas por esses SNPs estão envolvidas na contração muscular, metabolismo e defesa celular. Portanto, é possível inferir que o pH tem impacto nas características de qualidade da carne e que polimorfismos em MR e o GR levam a mudanças na atividade do eixo HPA, no perfil metabólico do organismo e no perfil proteico do músculo, sugerindo que esses genes estão envolvidos em uma complexidade de funções e podendo ser alvos de estudos em sistemas de produção que visam melhorar a produtividade. / The hypothalamic-pituitary-adrenal axis is the main neuroendocrine system involved in the regulation and adaptation in stress response and the primary hormone secreted is cortisol. Cortisol exerts its effects through the mineralocorticoid (MR) and glucocorticoid (GR) receptors. Variations in the genes of these receptors have been associated with sensitivity to glucocorticoids and changes in the metabolic profile. The general objective of this work was to understand the variability in relation to physiological responses of cattle through identification of genetic polymorphisms in genes involved in stress response and, checking the consequences of this genetic variation in meat quality traits. Thus, three approaches have been proposed: (1) evaluate the incidence of DFD meat (dark, firm and dry) and its impact on metabolics, endocrines profiles and meat quality traits, since stress is the major factor that lead to this unfavorable condition; (2) evaluate the contribution of genetic factors through identification single nucleotide polymorphisms (SNPs) in the MR and GR gene and its association with the measured traits; (3) evaluate the effects of these polymorphisms on the protein profile of bovine muscle. A total of 241 Nellore cattle were used. The results evidenced direct implications of 24 hours pH post-mortem in color attributes and cooking losses. The incidence of DFD meat (pH >= 5.8) was 18.7%. The polymorphisms identified demonstrated to influence some on measured characteristics. The NR3C2_1 and NR3C2_2 SNPs in MR gene were associated with muscle glycogen content and post-mortem adrenocorticotropic hormone (ACTH) plasma levels and, the NR3C1_1 SNP in GR was associated with post-mortem cortisol plasma levels. The proteomic analysis demonstrated that most proteins regulated by these SNPs are involved in muscle contraction, metabolism and cellular defense. Therefore, it is possible to infer that pH has impact on meat quality traits and MR and GR polymorphisms lead to changes in the HPA axis activity, metabolic profile and protein muscle profile, suggesting that these genes are involved in a complexity of functions and may be targets for studies on production systems to improve productivity.

O colágeno XVIII é um componente das membranas basais com diversos domínios funcionais, como a endostatina e o domínio frizzled, que têm importante papel em processos celulares como proliferação e diferenciação. COL18A1 possui dois promotores alternativos: o promotor 1, que regula a síntese da variante NC11-303, e o promotor 2 responsável pelas variantes NC11- 728 e NC11-493, expressas por hepatócitos. Existe uma variação interindividual da endostatina circulante e da expressão do colágeno XVIII no fígado. A expressão do colágeno XVIII/endostatina foi correlacionada com a progressão tanto do hepatocarcinoma (HCC), quanto da fibrose/cirrose hepática. Elucidar a regulação da expressão de COL18A1 pode auxiliar na compreensão dessa variação interindividual e da progressão dessas doenças. Neste trabalho demos início a caracterização do promotor 2 do COL18A1. Identificamos na seqüência predita como promotora cinco regiões conservadas entre humanos e camundongos. A análise in silico e funcional dessas regiões revelou que os fatores de transcrição, Sp1, Sp3, YY1, Oct-1, C/EBPα e C/EBPβ, interagem com as mesmas. Demonstramos que C/EBPβaumenta a taxa de transcrição do promotor 2 em hepatócitos, e que existe uma correlação positiva da expressão de NC11-493 com C/EBPαe C/EBPβem tecido hepático cirrótico e tumoral. As expressões de C/EBPαem tecido hepático cirrótico e tumoral estão diretamente correlacionadas, enquanto que os níveis de NC11-493 nos tumores estão inversamente correlacionados com o tamanho dos mesmos. Mostramos a existência de diversos SNPs no promotor 2. O SNP-700T/G, funcional in vitro, afeta a interação de Sp3 e YY1 com essa região regulatória. A deleção da região do SNP indicou que ela possui elementos importantes para a transcrição em hepatócitos, apesar deste SNP não estar relacionado com o nível de expressão do colágeno XVIII em fígado fibrótico ou com susceptibilidade a HCC. O SNP- 700T/G está em desequilíbrio de ligação com o SNPc.1135C/T, no domínio frizzled do colágeno XVIII. Não foi possível elucidar a funcionalidade do SNPs c.1135C/T in vitro, mas os haplótipos formados por esses dois SNPs têm diferentes frequências entre descendentes de europeus e de africanos. Nosso trabalho traz importantes contribuições e abre novas perspectivas para a compreensão da regulação do colágeno XVIII em fígado humano, tanto em situações fisiológicas, quanto em processos fibrogênicos e tumorigênicos¶ / Collagen XVIII is a basal membrane component with several funcional domains, such as endostatin and frizzled domains, which have important roles in cellular processes such as proliferation and differentiation. COL18A1 has two promoter regions: promoter 1, that regulates the synthesis of NC11-303 isoform, and promoter 2, localized in intron 2, responsible for NC11-728 and NC11-493 isoforms expressed by hepatocytes. There is a large interindividual variation in circulating endostatin and in collagen XVIII liver expression. Collagen XVIII/endostatin levels were correlated with hepatocellular carcinoma (HCC) progression, as well as liver fibrosis/cirrhosis, conditions that precede HCC. Elucidating the mechanisms that regulate COL18A1 expression in hepatocytes may help understanding its variation among individuals and liver disease stages, as well as contribute to new treatment strategies. In this work we began to characterize COL18A1 promoter region 2. We identified in the predicted promoter sequence five conserved regions between human and mouse. The in silico and functional analysis of these regions revealed that transcription factors Sp1, Sp3, YY1, Oct-1, C/EBPα and C/EBPβ interact with them. We have demonstrated that C/EBPβ increases promoter 2 transcription rate in hepatocytes, and that there is a positive correlation of NC11-493 expression with that of C/EBPα and C/EBPβ in cirrhotic and tumor liver samples. Non-tumor and tumor C/EBPα expressions positively correlate between themselves, while NC11-493 tumor expression inversely correlates with tumor size. We also showed that there are several SNPs in COL18A1 promoter 2 region. SNP-700T/G, functional in vitro, affects Sp3 and YY1 interaction with the promoter 2 region and deletion of the SNP region indicated that this sequence has important hepatocyte regulatory elements. Our results suggest that this SNP does not significantly affects COL18A1 expression in fibrotic/cirrhotic liver and is not associated with HCC susceptibility. SNP-700T/G is in linkage disequilibrium with SNPc.1135C/T, at collagen XVIII frizzled domain. We could not elucidate SNPc.1135C/T functionality in vitro, but the haplotypes formed by these two SNPs have different frequencies in European and African descendants. In conclusion, our work brings important contributions and opens new perspectives for the comprehension of collagen XVIII regulation in human liver in physiological situations, as well as in fibrotic/cirrhotic and tumorigenic process.

Polimorfismos em genes de receptores Toll-like (TLR) podem modular o risco de desenvolvimento de infecção, inflamação crônica e câncer. O objetivo deste estudo foi investigar a associação de polimorfismos em TLR ao risco aumentado de desenvolvimento de câncer de cabeça e pescoço, o carcinoma epidermóide (CEC) de boca e de laringe, e lesões bucais com potencial de transformação maligna, como o líquen plano oral (LPO), incluindo lesões idiopáticas e lesões liquenóides (LLO). Para tal foi conduzido um estudo caso-controle com 40 pacientes com CEC de boca, 35 com CEC de laringe, 175 com LPO (129 idiopático e 46 LLO) e 89 controles saudáveis, todos de origem basca. Oito SNP nos TLR1, TLR2, TLR4, TLR6, TLR9 e TLR10 foram genotipados por ensaios TaqMan® ou pirosequenciamento. A análise estatística por meio do teste qui-quadrado mostrou que a variante A, para o SNP TLR2-rs4696480 aumentou significativamente o risco para o desenvolvimento de CEC de boca (p=0.03) e LLO (p=0.0223). O genótipo AT representa risco de desenvolvimento de CEC de boca aumentado em 5.3 vezes quando comparado ao genótipo TT (OR=5.3, IC95%=1.19-13.63), e genótipo AA em 6.6 vezes (OR=6.6, IC95%=1.30-33.89). Quanto ao desenvolvimento de LLO, o genótipo AT representa um aumento no risco de 4.6 vezes comparado ao genótipo TT (OR=4.6, IC95%=1.55-13.38) e o genótipo AA em 4.1 vezes (OR=4.1, IC95%=1.33-12.88). Embora os genótipos AT e AA ocorram com significativa frequência no grupo LPO idiopático (p=0.045), este SNP não foi correlacionado estatisticamente à susceptibilidade de desenvolvimento deste. O SNP TLR2-rs4696480 pode ser relevante para o risco de desenvolvimento de CEC de boca e LLO nesta população, incentivando novos estudos sobre a possível associação destes grupos de doenças e SNP no gene do TLR2, colaborando com a demonstração de polimorfismos de TLR como marcadores úteis do prognóstico e prevenção do câncer de boca. / Polymorphisms in toll-like receptor (TLR) genes may modulate the risk of infection, chronic inflammation and cancer. This study investigated whether TLR polymorphisms were associated with an increased risk of head and neck cancer, including oral (OSCC) and laryngeal squamous cell carcinoma (LSCC); and oral premalignant disorders such as oral lichenoid disease (OLD), including oral lichen planus (OLP) and oral lichenoid lesions (OLL). This case-control study included 40 OSCC, 35 LSCC, 175 OLD (129 OLP and 46 OLL) patients and 89 healthy controls, all of them from the Basque Country. Genetic polymorphisms in TLR1, TLR2, TLR4, TLR6, TLR9, and TLR10 were genotyped by TaqMan® assays or pyrosequencing. Chi-square analysis showed that the variant A for the SNP TLR2-rs4696480 increased OSCC (p=0.03) and OLL (p=0.0223) risk significantly. AT genotype increases the risk of developing an OSCC by 5.3 times compared with TT genotype (OR=5.3, 95%CI=1.19-13.63), and the AA by 6.6 times (OR=6.6, 95%CI=1.30-33.89). AT genotype increases the risk of developing OLL by 4.6 times compared with TT genotype (OR=4.6, 95%CI=1.55-13.38) and the AA by 4.1 times (OR=4.1, 95%CI=1.33-12.88). Although these mutated genotypes were significantly frequent in the OLP group (p=0.045), this SNP was not correlated with OLP susceptibility. TLR2-rs4696480 polymorphism may be relevant to OSCC and OLL susceptibility in this population; encouraging further studies to assess the possible association of this group of potentially malignant disorders and oral cancer with TLR2 SNP, which may help to demonstrate that TLR polymorphisms may be useful markers to prognosis and cancer prevention.

Depuis le premier séquençage du génome humain au début des années 2000, de grandes initiatives se sont lancé le défi de construire la carte des variabilités génétiques inter-individuelles, ou bien encore celle des altérations de l'ADN tumoral. Ces projets ont posé les fondations nécessaires à l'émergence de la médecine de précision, dont le but est d'intégrer aux dossiers médicaux conventionnels les spécificités génétiques d'un individu, afin de mieux adapter les traitements et les stratégies de prévention. La traduction des variations et des altérations de l'ADN en prédictions phénotypiques constitue toutefois un problème difficile. Les séquenceurs ou puces à ADN mesurent plus de variables qu'il n'y a d'échantillons, posant ainsi des problèmes statistiques. Les données brutes sont aussi sujettes aux biais techniques et au bruit inhérent à ces technologies. Enfin, les vastes réseaux d'interactions à l'échelle des protéines obscurcissent l'impact des variations génétiques sur le comportement de la cellule, et incitent au développement de modèles prédictifs capables de capturer un certain degré de complexité.Cette thèse présente de nouvelles contributions méthodologiques pour répondre à ces défis.Tout d'abord, nous définissons une nouvelle représentation des profils de mutations tumorales, qui exploite leur position dans les réseaux d'interaction protéine-protéine. Pour certains cancers, cette représentation permet d'améliorer les prédictions de survie à partir des données de mutations, et de stratifier les cohortes de patients en sous-groupes informatifs. Nous présentons ensuite une nouvelle méthode d'apprentissage permettant de gérer conjointement la normalisation des données et l'estimation d'un modèle linéaire. Nos expériences montrent que cette méthode améliore les performances prédictives par rapport à une gestion séquentielle de la normalisation puis de l'estimation. Pour finir, nous accélérons l'estimation de modèles linéaires parcimonieux, prenant en compte des interactions deux à deux, grâce à un nouvel algorithme. L'accélération obtenue rend cette estimation possible et efficace sur des jeux de données comportant plusieurs centaines de milliers de variables originales, permettant ainsi d'étendre la portée de ces modèles aux données des études d'associations pangénomiques. / Since the first sequencing of the human genome in the early 2000s, large endeavours have set out to map the genetic variability among individuals, or DNA alterations in cancer cells. They have laid foundations for the emergence of precision medicine, which aims at integrating the genetic specificities of an individual with its conventional medical record to adapt treatment, or prevention strategies.Translating DNA variations and alterations into phenotypic predictions is however a difficult problem. DNA sequencers and microarrays measure more variables than there are samples, which poses statistical issues. The data is also subject to technical biases and noise inherent in these technologies. Finally, the vast and intricate networks of interactions among proteins obscure the impact of DNA variations on the cell behaviour, prompting the need for predictive models that are able to capture a certain degree of complexity. This thesis presents novel methodological contributions to address these challenges. First, we define a novel representation for tumour mutation profiles that exploits prior knowledge on protein-protein interaction networks. For certain cancers, this representation allows improving survival predictions from mutation data as well as stratifying patients into meaningful subgroups. Second, we present a new learning framework to jointly handle data normalisation with the estimation of a linear model. Our experiments show that it improves prediction performances compared to handling these tasks sequentially. Finally, we propose a new algorithm to scale up sparse linear models estimation with two-way interactions. The obtained speed-up makes this estimation possible and efficient for datasets with hundreds of thousands of main effects, thereby extending the scope of such models to the data from genome-wide association studies.

Polimorfismos em genes de receptores Toll-like (TLR) podem modular o risco de desenvolvimento de infecção, inflamação crônica e câncer. O objetivo deste estudo foi investigar a associação de polimorfismos em TLR ao risco aumentado de desenvolvimento de câncer de cabeça e pescoço, o carcinoma epidermóide (CEC) de boca e de laringe, e lesões bucais com potencial de transformação maligna, como o líquen plano oral (LPO), incluindo lesões idiopáticas e lesões liquenóides (LLO). Para tal foi conduzido um estudo caso-controle com 40 pacientes com CEC de boca, 35 com CEC de laringe, 175 com LPO (129 idiopático e 46 LLO) e 89 controles saudáveis, todos de origem basca. Oito SNP nos TLR1, TLR2, TLR4, TLR6, TLR9 e TLR10 foram genotipados por ensaios TaqMan® ou pirosequenciamento. A análise estatística por meio do teste qui-quadrado mostrou que a variante A, para o SNP TLR2-rs4696480 aumentou significativamente o risco para o desenvolvimento de CEC de boca (p=0.03) e LLO (p=0.0223). O genótipo AT representa risco de desenvolvimento de CEC de boca aumentado em 5.3 vezes quando comparado ao genótipo TT (OR=5.3, IC95%=1.19-13.63), e genótipo AA em 6.6 vezes (OR=6.6, IC95%=1.30-33.89). Quanto ao desenvolvimento de LLO, o genótipo AT representa um aumento no risco de 4.6 vezes comparado ao genótipo TT (OR=4.6, IC95%=1.55-13.38) e o genótipo AA em 4.1 vezes (OR=4.1, IC95%=1.33-12.88). Embora os genótipos AT e AA ocorram com significativa frequência no grupo LPO idiopático (p=0.045), este SNP não foi correlacionado estatisticamente à susceptibilidade de desenvolvimento deste. O SNP TLR2-rs4696480 pode ser relevante para o risco de desenvolvimento de CEC de boca e LLO nesta população, incentivando novos estudos sobre a possível associação destes grupos de doenças e SNP no gene do TLR2, colaborando com a demonstração de polimorfismos de TLR como marcadores úteis do prognóstico e prevenção do câncer de boca. / Polymorphisms in toll-like receptor (TLR) genes may modulate the risk of infection, chronic inflammation and cancer. This study investigated whether TLR polymorphisms were associated with an increased risk of head and neck cancer, including oral (OSCC) and laryngeal squamous cell carcinoma (LSCC); and oral premalignant disorders such as oral lichenoid disease (OLD), including oral lichen planus (OLP) and oral lichenoid lesions (OLL). This case-control study included 40 OSCC, 35 LSCC, 175 OLD (129 OLP and 46 OLL) patients and 89 healthy controls, all of them from the Basque Country. Genetic polymorphisms in TLR1, TLR2, TLR4, TLR6, TLR9, and TLR10 were genotyped by TaqMan® assays or pyrosequencing. Chi-square analysis showed that the variant A for the SNP TLR2-rs4696480 increased OSCC (p=0.03) and OLL (p=0.0223) risk significantly. AT genotype increases the risk of developing an OSCC by 5.3 times compared with TT genotype (OR=5.3, 95%CI=1.19-13.63), and the AA by 6.6 times (OR=6.6, 95%CI=1.30-33.89). AT genotype increases the risk of developing OLL by 4.6 times compared with TT genotype (OR=4.6, 95%CI=1.55-13.38) and the AA by 4.1 times (OR=4.1, 95%CI=1.33-12.88). Although these mutated genotypes were significantly frequent in the OLP group (p=0.045), this SNP was not correlated with OLP susceptibility. TLR2-rs4696480 polymorphism may be relevant to OSCC and OLL susceptibility in this population; encouraging further studies to assess the possible association of this group of potentially malignant disorders and oral cancer with TLR2 SNP, which may help to demonstrate that TLR polymorphisms may be useful markers to prognosis and cancer prevention.

The Human Genome Project, initiated in 1990, creates an enormous amount of excitement in human genetics—a field of study that seeks answers to the understanding of human evolution, diseases and development, gene therapy, and preventive medicine. The first completion of a human genome in 2003 and the breakthroughs of sequencing technologies in the past few years deliver the promised benefits of genome studies, especially in the roles of genomic variability and human health. However, intensive resource requirements and the associated costs make it infeasible to experimentally verify the effect of every genetic variation. At this stage of genome studies, in silico predictions play an important role in identifying putative functional variants. The most common practice for genome variant evaluation is based on the evolutionary conservation at the mutation site. Nonetheless, sequence conservation is not the absolute predictor for deleteriousness since phylogenetic diversity of aligned sequences used to construct the prediction algorithm has substantial effects on the analysis. This dissertation aims at overcoming the weaknesses of the conservation-based assumption for predicting the variant effects. The dissertation describes three different integrative computational approaches to identify a subset of high-priority amino acid mutations, derived from human genome data. The methods investigate variant-function relationships in three aspects of genome studies—personal genomics, genomics of epilepsy disorders, and genomics of variable drug responses. For genetic variants found in genomes of healthy individuals, an eight-level variant classification scheme is implemented to rank variants that are important towards individualized health profiles. For candidate genetic variants of epilepsy disorders, a novel 3-dimensional structure-based assessment protocol for amino acid mutations is established to improve discrimination between neutral and causal variants at less conserved sites, and to facilitate variant prioritization for experimental validations. For genomic variants that may affect inter-individual variability in drug responses, an explicit structure-based predictor for structural disturbances is developed to efficiently evaluate unknown variants in pharmacogenes. Overall, the three integrative approaches provide an opportunity for examining the effects of genomic variants from multiple perspectives of genome studies. They also introduce an efficient way to catalog amino acid variants on a large scale genome data.

The aim of the thesis – to investigate the role of the inflammatory bowel disease associated genetic variants in a subset of Crohn’s disease and ulcerative colitis patients from Lithuania and Latvia and to test the relation of genetic markers to disease phenotype. The following objectives were accomplished: the associations of the inflammatory bowel disease associated single nucleotide polymorphisms in the subset of Crohn’s disease and ulcerative colitis patients were determined; the associations of the single nucleotide polymorphisms with the phenotype of the inflammatory bowel disease were evaluated; the interactions of single nucleotide polymorphisms (SNP-SNP) and their association with inflammatory bowel disease were determined; the significance of the combinations of disease associated single nucleotide polymorphisms for diagnosis of inflammatory bowel disease was evaluated. TaqMan and SNPlex genotyping methods were used in this work. The statistical data analysis consisted of: statistical study power and data quality evaluations; single marker case-control association analysis using χ2 or Fisher's exact tests, Breslow-Day test, Cochran-Mantel-Haenzsel test and post-hoc Bonferroni correction; genotype–phenotype association analysis using χ2 test and post-hoc Bonferroni correction; SNP-SNP epistasis for case-control sample using logistic regression test and post-hoc Bonferroni correction; In sicilo prediction of gene interactive network; genetic risk profile construction... [to full text] / Disertacijos tikslas – ištirti su uždegiminėmis žarnyno ligomis siejamų ge¬netinių žymenų reikšmę Krono liga bei opiniu kolitu sergantiems Lietuvos ir Latvijos ligoniams bei nustatyti žymenų ryšį su ligos fenotipu. Tikslui pasiekti, įgyvendinti šie uždaviniai: ištirta su uždegiminėmis žarnyno ligomis siejamų vieno nukleotido polimorfizmų sąsaja su Krono liga bei opiniu kolitu; nustatyta vieno nukleotido polimorfizmų sąsaja su uždegiminių žarny¬no ligų fenotipu; ištirtos vieno nukleotido polimorfizmų tarpusavio epistazinės sąvei¬kos (VNP-VNP) bei jų ryšys su uždegiminėmis žarnyno ligomis; nustatytas su liga siejamų vieno nukleotido polimorfizmų derinių reikšmingumas uždegiminių žarnyno ligų diagnostikai. Darbe taikyti SNPlex ir TaqMan genotipavimo metodai. Statistinę duomenų analizę sudarė: studijos galios bei duomenų kokybės įvertinimas; atvejo-kontrolės genų sąsajos tyrimas, taikant χ2 arba Fišerio kriterijus, Breslow-Day testą, Cochran-Mantel-Haenzsel testą bei post-hoc Bonferroni kriterijų; genų sąsajų su Krono ligos ir opinio kolito fenotipu tyrimai, naudojant χ2 bei post-hoc Bonferroni kriterijų; VNP-VNP epistazinės sąveikos įvertinimas, taikant logistinės regresijos testą bei post-hoc Bonferroni kriterijų; genų tarpusavio sąveikos tinklo in silico tyrimas; genetinės rizikos profilio sudarymas, naudojant logistinę regresinę analizę.